Effects of microgravity on vestibular development and function in rats: genetics and environment

Our anatomical and behavioral studies of embryonic rats that developed in microgravity suggest that the vestibular sensory system, like the visual system, has genetically mediated processes of development that establish crude connections between the periphery and the brain. Environmental stimuli also regulate connection formation including terminal branch formation and fine-tuning of synaptic contacts. Axons of vestibular sensory neurons from gravistatic as well as linear acceleration receptors reach their targets in both microgravity and normal gravity, suggesting that this is a genetically regulated component of development. However, microgravity exposure delays the development of terminal branches and synapses in gravistatic but not linear acceleration-sensitive neurons and also produces behavioral changes. These latter changes reflect environmentally controlled processes of development.     

Identification of novel multi-transmembrane proteins from genomic databases using quasi-periodic structural properties

MOTIVATION: Identification of novel G protein-coupled receptors and other multi-transmembrane proteins from genomic databases using structural features. RESULTS: Here we describe a new algorithm for identifying multi-transmembrane proteins from genomic databases with a specific application to identifying G protein-coupled receptors (GPCRs) that we call quasi-periodic feature classifier (QFC). The QFC algorithm uses concise statistical variables as the 'feature space' to characterize the quasi-periodic physico-chemical properties of multi-transmembrane proteins. For the case of identifying GPCRs, the variables are then used in a non-parametric linear discriminant function to separate GPCRs from non-GPCRs. The algorithm runs in time linearly proportional to the number of sequences, and performance on a test dataset shows 96% positive identification of known GPCRs. The QFC algorithm also works well with short random segments of proteins and it positively identified GPCRs at a level greater than 90% even with segments as short as 100 amino acids. The primary advantage of the algorithm is that it does not directly use primary sequence patterns which may be subject to sampling bias. The utility of the new algorithm has been demonstrated by the isolation from the Drosophila genome project database of a novel class of seven-transmembrane proteins which were shown to be the elusive olfactory receptor genes of Drosophila.     

Bacterial chromosomal painting for in situ monitoring of cultured marine bacteria

We previously described a new method, bacterial chromosomal painting (BCP), for the in situ identification of bacterial cells. Here, we describe the application of this technique to study the ecology and physiology of cultured marine pelagic bacteria from the western Sargasso Sea (WSS). A total of 86 bacteria were isolated from seawater collected from near the surface, at a depth of 250 m and from nutrient-amended seawater incubations. The 10 bacterial isolates that were best represented in environmental genomic DNA from the WSS were selected using reverse genome probing. BCP hybridization cell counts were used to determine the depth-specific distribution of one of the alpha proteobacterial isolates, B5-6, in the WSS during two thermal stratification regimes: stratified and partially mixed. The maximum cell count measured for B5-6 at the summer deep chlorophyll maximum was approximately 4% of the total cell count. This study is the first application of BCP to natural environments .     

Responses of Nine Trifolium repens L. Populations to Ultraviolet-B Radiation: Differential Flavonol Glycoside Accumulation and Biomass Production

This study aimed to quantify and identify flavonoids involvedin the response of nine populations of white clover (Trifoliumrepens L.) to ultraviolet-B radiation (UV-B). Plants were grownfor 12 weeks in controlled environment rooms with or withoutsupplemental UV-B radiation of 13.3 kJ m^-2d^-1. Methanol–waterextractable flavonoids were quantified using high performanceliquid chromatography (HPLC). Two major peaks showed significantenhancement in the HPLC chromatogram in response to supplementalUV-B. The structures of the compounds responsible were identifiedby¹H and¹³C nuclear magnetic resonance (NMR) spectroscopy tobe the flavonols quercetin-3-O-ß- D -xylopyranosyl-(1 2)-ß- D -galactopyranoside and kaempferol-3-O-ß-D -xylopyranosyl-(1 2)-ß- D -galactopyranoside. Withsupplemental UV-B, quercetin glycoside levels increased on averageby 200% while the kaempferol glycoside response was much smaller.Significant differences in flavonol accumulation were foundamong T. repens populations, both constitutively and in responseto UV-B. Stress-adapted populations displayed particularly highflavonol levels under UV-B. There was an inverse correlationbetween plant productivity and quercetin accumulation. Furthermore,higher quercetin accumulation under UV-B was correlated withtolerance against UV-B-induced growth reduction. In conclusion,within-species comparisons in T. repens lend support to a distinctrole for ortho -dihydroxylated flavonoids in the adaptationto UV-B stress and suggest particular advantages in this UV-B-inducedbiochemical adaptation for populations characterized by lowhabitat and plant productivity. Copyright 2000 Annals of BotanyCompany Ultraviolet-B, Trifolium repens, white clover, HPLC, NMR, flavonoids, flavonols, quercetin, kaempferol, biomass, genetic variation, intraspecific     

The effect of T1 and T2 cytokines on the cytotoxic T cell response to mannan-MUC1

MUC1 is a mucin over-expressed in breast cancer and a proposed target for immunotherapy. By immunising mice with MUC1 conjugated to mannan (M-FP), CD8⁺ MHC-class-I restricted cytotoxic T lymphocytes (CTL), of high CTL precursor (CTLp) frequency (1/8000) and with significant tumour protection, can be induced. The effect of various cytokines [interleukin-2 (IL-2), IL-4, IL-6, IL-7, interferon γ (IFNγ), and granulocyte/macrophage-colony-stimulating factor (GM-CSF)] on the MUC1 CTL immune response was investigated (a) by measuring the frequencies of CTLp in mice immunised with vaccinia virus constructs containing recombinant cytokines and M-FP, or (b) by immunising cytokine- or cytokine-receptor-knockout (−/−) mice with M-FP. Vaccinia virus (VV) constructs containing recombinant cytokines were used either individually or in combination in vivo with M-FP immunisation. M-FP immunisations combined with VV-IL-2, VV-IL-7 and VV-GM-CSF, and combinations of VV-IFNγ + VV-IL-2, VV-IFNγ + VV-IL-4 or VV-GM-CSF + VV-IL-7 increased CTLp frequencies up to threefold (1/17 666: M-FP + VV-GM-CSF + VV-IL-7) compared to M-FP (1/77 500) alone. By contrast, M-FP combined with VV-IL-4 decreased the CTLp frequency threefold whereas VV-IL-6 and VV-IFNγ had no effect. Studies in cytokine- and cytokine-receptor-gene-knockout (−/−) mice demonstrated that mice that are IL-2 −/− and IL-7 receptor −/− produce the same CTLp response to M-FP as do control mice, whereas responses in the IL-6 −/−, IL-10 −/− and IFNγ−/− mice were marginally improved and responses to M-FP in IL-4 −/− and tumour necrosis factor receptor 2 −/− mice were weaker. In spite of the increase in CTLp frequency, this was not reflected in an in vivo tumour model. Tumour challenges using MUC1⁺ P815 cells, demonstrated that the addition of cytokines had little additive effect on the already effective tumour-regression capabilities of M-FP alone. Received: 24 September 1998 / Accepted: 21 September 1999     

Cytochromes P450 involved with benzene metabolism in hepatic and pulmonary microsomes

Benzene is an occupational hazard and environmental toxicant found in cigarette smoke, gasoline, and the chemical industry. The major health concern associated with benzene exposure is leukemia. The toxic effects of benzene are dependent on its metabolism by the cytochrome P450 enzyme system. Previous research has identified CYP2E1 as the primary P450 isozyme responsible for benzene metabolism at low concentrations, whereas CYP2B1 is involved at higher concentrations. Our studies using microsomal preparations from human, mouse, and rat indicate that CYP2E1 is the P450 isozyme primarily responsible for benzene metabolism in lung and in liver. CYP2B isozymes have little involvement in benzene metabolism by either lung or liver. Our results also indicate that isozymes of the CYP2F subfamily may play a role in benzene metabolism by lung.     

Development of improved high-performance liquid chromatography conditions for nonisotopic detection of isoaspartic acid to determine the extent of protein deamidation

A rapid and accurate ion-pairing reversed-phase high-performance liquid chromatography (IP-RP-HPLC) procedure has been developed for nonisotopic detection of isoaspartic acid residues in protein or peptides resulting from deamidation of asparagine residues. The IP-RP-HPLC procedure specifically detects and quantifies S-adenosylhomocysteine (SAH). SAH is a by-product of the reaction between protein isoaspartyl methyltransferase (PIMT), S-adenosylmethionine (SAM), and isoaspartic acid residues. The HPLC conditions described in this paper have been demonstrated to offer significantly better reproducibility compared to earlier studies. The HPLC method allows determination of the extent of protein deamidation without the use of radioisotopes and therefore offers significant advantages for biopharmaceutical development laboratories.     

Too much interference: injection of double-stranded RNA has nonspecific effects in the zebrafish embryo

We have investigated the ability of double-stranded RNA (dsRNA) to inhibit gene expression in a vertebrate, the zebrafish, Danio rerio. Injection of dsRNA corresponding to the T-box gene tbx16/spadetail (spt) into early wild-type embryos caused a rapid and dramatic loss of tbx16/spt mRNA in the blastula. mRNAs from the papc, tbx6, and gata1 genes, which depend on tbx16/spt function for their expression, were reduced, apparently mimicking the spt mutant phenotype. However, mRNAs from a number of genes that are unaffected by the spt mutation, such as beta catenin, stat3, and no tail, were also lost, indicating that the "interference" effect of tbx16/spt dsRNA was not restricted to the endogenous tbx16/spt mRNA. We compared the effects of injecting dsRNA from the zebrafish tbx16/spadetail, nieuwkoid/bozozok, and Brachyury/no tail genes with dsRNA from the bacterial lacZ gene. In each case the embryos displayed a variable syndrome of abnormalities at 12 and 24 h postfertilization. In blind studies, we could not distinguish between the effects of the various dsRNAs. Consistent with a common effect of dsRNA, regardless of sequence, injection of dsRNA from the lacZ gene was likewise effective in strongly reducing tbx16/spt and beta catenin mRNA in the blastula. These findings indicate that, despite published reports, the current methodology of double-stranded RNA interference is not a practical technique for investigating zygotic gene function during early zebrafish development.     

Molecular evolution of odorant-binding protein genes OS-E and OS-F in Drosophila

The Drosophila olfactory genes OS-E and OS-F are members of a family of genes that encode insect odorant-binding proteins (OBPs). OBPs are believed to transport hydrophobic odorants through the aqueous fluid within olfactory sensilla to the underlying receptor proteins. The recent discovery of a large family of olfactory receptor genes in Drosophila raises new questions about the function, diversity, regulation, and evolution of the OBP family. We have investigated the OS-E and OS-F genes in a variety of Drosophila species. These studies highlight potential regions of functional significance in the OS-E and OS-F proteins, which may include a region required for interaction with receptor proteins. Our results suggest that the two genes arose by an ancient gene duplication, and that in some lineages, one or the other gene has been lost. In D. virilis, the OS-F gene shows a different spatial pattern of expression than in D. melanogaster. One of the OS-F introns shows a striking degree of conservation between the two species, and we identify a putative regulatory sequence within this intron. Finally, a phylogenetic analysis places both OS-E and OS-F within a large family of insect OBPs and OBP-like proteins.